175

Biochimico et Biophysics Acta, 450 (1976) 175-184 @ Elsevier/North-Holland Biomedical Press

BBA 56865

PROPERTIES HEPATOMA

OF MICROSOMAL

PHOSPHOLIPASES

IN RAT LIVER AND

ROGER H. LUMB and KAREN F. ALLEN From the Department (U.S.A.)

of Biology, Western Carolina University, Cullowhee, N.C. 28723

(Received May 12th, 1976)

Summary Phospholipase A,, AZ and lysophospholipase activities in microsomes of Novikoff hepatoma, host rat liver and regenerating rat liver were compared using l-[ 9’,10’-3HZ]palmitoyl-2-[ 1 ‘-14C] linoleoyl-sn-glycero-3-phosphoethanoll-[ 1’-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and amine, l-[9’,10’-3H~]palmitoyl-sn-glycero-3-phosphoeth~ol~ine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca” for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca” for activity at both pH values. 3. Phospholipase A, activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A, activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using l[ 1’-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2’ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.

Introduction The microsomal fraction for de novo phospholipid

of rat liver has been shown to contain the enzymes synthesis, while other membranes of the cell are

176

dependent upon the endoplasmic reticulum for their supply of phospholipids [ 1,2]. By their participation in the deacylation-reacylation cycle microsomal phospholipases have a role in modifying the phospholipid composition of cell membranes [3], and alterations in the enzyme activities could directly affect membrane function [ 41. It is becoming increasingly apparent that membrane phospholipids have a direct role in cell surface receptor function [5] and in growth stimulation [6,7]. Membranes of neoplastic cells show a number of differences in comparison to normal cells [S], and several papers have appeared reporting differences in membrane phospholipid content [g-13]. Alterations in phospholipid metabolism have also been implicated in the failure of neoplastic cells to form communicating intercellular junctions [ 14-161, and in hepatocarcinogen challange [17]. Membrane-bound phospholipases may have a role in determining some of the altered characteristics of membranes from neoplastic cells. A number of investigators have examined phospholipase activities in rat liver subcellular fractions (for reviews see refs. 1,3,18). It has been established that phospholipase A, (EC 3.1.1.32) and phospholipase A2 (EC 3.1.1.4) activities are associated with microsomes [ 19-221 and lysosomes [23,24], phospholipase AZ activity with mitochondria [ 25-311, while plasma membranes show as yet undefined specificities [ 32,261. Lysophospholipase activity is associated with most subcellular fractions [ 181. A clear picture of the pH optima, effect of calcium and the effects of detergents on phospholipases in animal tissues has not yet emerged. We have investigated microsomal phospholipase A,, A2 and lysophospholipase activities in normal rat liver, regenerating rat liver and Novikoff hepatoma as an initial step in the clarification of the role these enzymes might play in determining the altered membrane characteristics of cancer cells. Materials and Methods Materials. [9,10-3H,]Palmitic acid and [ l-14C]linoleic acid were obtained from New England Nuclear. The sodium boro[3H] hydride was obtained from Amersham/Searle Corporation, and the Cro talus adaman teus venom from Sigma Corporation. Preparation of microsomes. Novikoff hepatomas were maintained by transplantation in Holtzman rats; the hepatomas and host livers were collected and used for preparation of the microsomal fractions on the sixth day after intraperitoneal injection of a small piece of hepatoma. The tissues were homogenized in 0.25 M sucrose containing 1 mM EDTA (2 g tissue/5 ml) with five strokes of a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 15 000 X g for 15 min, the supernatant decanted and recentrifuged at 105 000 X g for 60 min. The microsomal pellets were washed once with 0.25 M sucrose containing 1 mM EDTA, resuspended in 0.25 M sucrose and used immediately. Purity of the microsomal preparations was determined by assay of acid phosphatase [37], succinate dehydrogenase [38], and NADPH: cytochrome c reductase [ 391. Small amounts of the suspensions were frozen for subsequent protein analysis by the method of Lowry et al. [40]. Partial hepatectomy was performed according to the procedure of Higgins and Anderson [41]. Regenerating livers were collected 24 h after partial hepa-

177

tectomy and microsomes obtained by the procedures described for host liver above. Preparation of substrates. The substrate, l-[ 3H]palmitoyl-2-acyl-sn-glycero-3phosphoethanolamine was prepared by incubation of [3H]palmitic acid with rat testis microsomes, which have been shown to incorporate saturated fatty acids specifically into the sn-1 position [42]. To obtain microsomes the testes were homogenized in 5 mM EDTA, 0.25 M sucrose (2 g/5 ml) and centrifuged at 15 000 X g for 15 min. The supernatant was then centrifuged at 105 000 X g for 60 min and the resulting microsomal pellet suspended in 0.25 M sucrose for immediate use. Microsomes from 0.5 g (wet weight) of testes and 90 nmol of [3H]palmitic acid were incubated in a 3 ml volume that contained 10 mM ATP, 0.1 mM CoA, 4 mM MgClz, and 0.1 M Tris - HCl at pH 7.1 for 1 h at 37°C. The neutral and phospholipids were separated on short silicic acid columns. Phosphatidylethanolamine was purified by thin-layer chromatography on silica gel H in 50 : 25 : 8 : 2 chloroform/methanol/acetic acid/water and 65 : 35 : 8 chloroform/methanol/ammonium hydroxide systems. The phosphatidylethanolamine was > 95% pure as judged by radioassay procedures; > 97% of the label was located at the sn-1 position as determined by Crotulus udumunteus venom phospholipase AZ hydrolysis [ 431. The 1-[9’,10’-3H,]palmitoyl-sn-glycero-3-phosphoethanolamine was prepared by treating the 1-[9’,10’-3H~]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine with Crotulus udumunteus venom [43]. The purity was > 95%. l-acyl-2-[ l’-14C] linoleoyl-sn-glycero-3-phosphoethanolamine was prepared by the procedures of Waite and van Deenen [13]. The biosynthesized phosphatidylethanolamine had a purity of > 95% with > 97% of the label in the sn-2 position. In all double-label experiments these two substrates were mixed to give appropriate 3H/‘4C ratios. Accumulation of [ 3H] lysophosphatidylethanolamine or [ “C]lysophosphatidylethanolamine are taken as minimum values for phospholipase AZ or phospholipase A1 activities, respectively. The method of Lumb and Snyder [ 441 was used to prepare l-[ l’-3H]hexadecyl-2-acyl-sn-glycerol-3-phosphoethanolamine from Ehrlich Ascites cells. The alk-l-enyl moiety was destroyed by treatment with HCl gas [45]. 34% of the phosphatidylethanolamine produced was in the alkyl acyl form [46]. More than 92% of the 3H was associated with the alkyl moiety. Incubation procedure. Substrates were suspended (100 nmol/0.4 ml) in water by sonication with a Sonic Dismembrator Model 150 (Artek Systems Corporation, 275 Adam Boulevard, Farmingdale, New York, 11735.) at 50 W. The preparation was held in a ice bath during two 30-s exposures interrupted by one 60-s period. The substrate was added to the incubation tubes immediately after addition of the microsomal preparation. All incubations were carried out in a final volume of 3 ml at 37°C. Bligh and Dyer [47] extractions were carried out on all samples; the extracted lipids were then stored in chloroform at -20°C. Thin layer chromatography was used to determine the products by using silica gel H 50 : 25 : 8 : 2 chloroform/methanol/acetic acid/water and silica gel G in 80 : 20 : 1 hexane/diethyl ether/acetic acid systems. Areas were scraped into a liquid scintillation mixture [48] and counted in a Packard Liquid Scintillation Spectrometer Model 3320. In some experiments, the lysophosphatidylethanlamine produced was identified by formation of the dinitrobenzene derivative followed by thin layer chromatography [49].

178

Results Assay of NADPH:cytochrome c reductase, succinate dehydrogenase and acid phosphatase activities in the cell free homogenate and microsomes shows that the preparations contained little mitochondrial or lysosomal contamination (Table I). Incubation of 1-acyl-2-[1’-‘4C]linoleoy1-s~-glycero-3-phosphoethanolamine with liver or hepatoma microsomes from pH 6 to pH 11 resulted in the release of labeled fatty acids at two pH optima (7.5 and 9.5). In both liver and hepatoma the hydrolysis required Ca*’ at pH 9.5, while at pH 7.5 only the hepatoma microsomes required Ca*’ for activity. The activities in hepatoma microsomes were linear for 2 h and up to 0.5 mg protein; in liver microsomes linearity was maintained for only 30 min and up to 0.8 mg protein. Increasing CaCl, concentration resulted in optimal activity at 5 mM; calcium concentrations above this value became inhibitory. Neither MgClz nor KC1 had any effect on the hydrolysis. Table II shows the results of incubating 1-[9’,10’-3H2]palmitoyl-2-[1’-14C]linoleoyl-sn-glycero-3-phosphoethanolamine with microsomes from host liver, and hepatoma. Host liver microsomes hydrolysed the substrate releasing equivalent amounts of both labeled fatty acids at pH 7.5 as well as at pH 9.5. The hydrolysis required Ca*’ at pH 9.5 but not at pH 7.5. Novikoff hepatoma microsomes also released both fatty acids at pH 7.5 and pH 9.5 but required Ca*’ for activity at both pH values. Addition of Triton X-100, which is known to inhibit many lysophospholipases, to the incubation mixtures resulted in the release of nearly stochiometric amounts of 2-[l ‘-14C]lino1eoyl-sn-glycero-3phosphoethanolamine and [3H]palmitic acid in host liver, while in Novikoff hepatoma microsomes addition of Triton X-100 inhibited all hydrolytic activity. The effect of increasing concentrations of Triton X-100 on the hydrolysis of phosphatidylethanolamine by host liver microsomes and Novikoff hepatoma microsomes is shown in Fig. 1. Addition of 0.5 mg Triton X-100 to the incubation mixtures inhibited all hydrolytic activity in both liver and hepatoma microsomes. Phospholipase Al activity was stimulated by increasing the concentration of Triton X-100 using liver microsomes; however, no such stimulation of hydrolytic activity was observed using hepatoma microsomes. Incubation of microsomes from regenerating rat liver showed hydrolytic patterns that were identical to those obtained using host liver microsomes.

TABLE

I

MARKER AND

ENZYME

HOST

ASSAYS

OF

MICROSOMAL

Percentage NADPH: Succinate Acid

PREPARATIONS

FROM

NOVIKOFF

HEPATOMA

LIVER

cytochrome

c reductase

dehydrogenase

Phosphatase

45

liver

Percentage

hepatoma

62

*

3

8

b

7 _

* Percent

of the

total

activity

in the homogenate

recovered

in the microsomal

fraction.

II BY MICROSOMES

OF NOVIKOFF

HEPTOMA

* [sHlFA,

(2) (7) (28) (22)

(5) (5) (8) (8) (0)

1 (1)

8 32 25 20

38 35 10 9 1

Liver

[%]linoleic

acid.

(nmol/mg protein per h) *

[sH]paImitic. acid; [%]FA,

pH 9.5 None Ca*+ Triton X-100 Ca’* + Triton X-100 Boiled

pH 7.5 None Ca’+ Triton X-100 Ca’+ + T&on X-100 Boiled

Additions

5 29 4 4 1

37 29 2 2 0

(2) (5) (4) (5) (0)

(4) (5) (1) (1) (0)

~__

5 33 1 2 0

4 15 2 1 0

(2) (4) (1) (1) (0)

(2) (3) (1) (1) (1)

Hepatoma

5 39 2 3 1

4 15 2 2 0

(3) (7) (2) (2) (0)

(3) (2) (1) (1) (1)

[ 14CJFA (C3HlLPE)

The incubation mixture contained 5 mM C&l 2. 0.05% Triton X-100, 0.1 M Tris . HCl (pH 7.5) or 0.1 glycine-NaOH (PH 9.5). 115 nmol of 1-[3HlpaImitoyl2-[14]linoleoylsn-3-phosphorylethanolamine and approximately 0.5 mg protein. Each value was obtained from the average of four results. The numbers in parentheses reflect the nmol of the corresponding lysophosphatidylethaokimine produced.

HYDROLYSIS OF 1-[3H]PALMITOYL-Z-[I~C]LINOLEOYL-sn-GLYCERO-3-PHOSPHORYLETHANOLAMINE AND HOST LIVER

TABLE

01

15 10 05 TRITON X- 100 (mg)

20

Fig. 1. The effect of increasing T&on X-100 concentration on the phospholipid hydrolytic activities of host rat liver microsomes (A) and Novikoff hepatoma microsomes (B) at pH 9.5. Incubation mixtures ’- 14CIlinoleoyl-sn-glycero-3-phosphoethanoin~, 0.5 included 115 nmol of l-[9’,10’-jH*lpalmitoyl-2-[l mg protein, 0.1 mM glycine-NaOH and 5 mM CaCl, in a total volume of 3 ml. Tubes were incubated 30 ‘H-lysophosphatidylethanolamine, l - - - - - -0, [‘%Imin at 37’C. A- - - - - -A, [ 3H] fatty acid, A[‘4Cllysophosphatidylethanolamine. fatty acid, oe,

Incubation of l-[ 1’-3H]hexadecyl-2-acyl-s~-glycero-3-phosphoethanolamine to detect phospholipase AZ activity with host liver and Novikoff hepatoma microsomes resulted in the production of l-[ 1’-3H] hexadecyl-sn-glycero-3phosphoethanolamine at pH 7.5 and pH 9.5. This activity required calcium at both pH values in both tissues, and was inhibited by Triton X-100 (Table III). When 1-[9’,lO’-3Hz]p~mitoyl-sn-glycero-3-phosphoeth~olamine was incubated with microsomes from host liver and hepatoma, labeled fatty acids were released at both pH 7.5 and pH 9.5. This hydrolytic activity was slightly inhibited by calcium and more extensively inhibited by Triton X-100 (Table III). The hydrolysis of phosphatidylethanolamine was completely inhibited by addition of 0.05% sodium dodecyl sulfate or 0.05% sodium deoxycholate in both liver and hepatoma at pH 9.5. Pre-incubation of the microsomes for 10 min at 45, 50, 55 and 60°C resulted in the loss of all hydrolytic activity at 55°C in both tissues at pH 9.5. Incubating increasing amounts of 1-[9’,10’-3H,]palmitoyl-2-[l’-14C]linoleo-

181

TABLE

III

PHOSPHOLIPASE A, AND LYSOPHOSPHOLIPASE HEPATOMA AND HOST LIVER The incubation mixture glycine-NaOH (PH 9.5).

ACTIVITIES

contained 5 mM CaCl,, 0.05% T&on 100 nmol substrate and approximately Additions

Enzyme

IN MICROSOMES

X-100, 0.1 M Tris 0.5 mg protein.

nmol/mg

protein

- HCl

(PH 7.5) or 0.1 mM

per h Hepatoma

Liver pH 9.5

pH 7.5

pH 9.5

X-100

1 5 1

3 20 3

1 10 1

3 52 6

X-100

30 59 12

44 41 17

126 99 6

92 73 2

pH 7.5 Phospholipase

OF NOVIKOFF

A2 * None Ca2+ Ca2++

Lysophospholipase

Triton

** None caZ+ C!a2+ + Triton

* l-[ 1’-sH]hexadecyl-2-acyl-sn-gIycero-3-phosphoethanolamine was used in nmol of 1-[l’-sHlhexadecyl-sn-glycerophosphoethanolamine released/mg protein per h. The values represent the average of four experiments. ** l-[9’,10’-3H]paImitoyl-sn-gIycero-3-phosphoethanolamine was used as substrate. Data is expressed in nmol of C3H]fatty acid released/mg protein per h. Each value is the average of two experiments.

yl-sn-glycero-3-phosphoethanolamine (50, 100, 150, 200, 250 and 300 nmol) produced decreasing percentages of hydrolysis and gave linear double-reciprocal plots for [3H]palmitic acid release. Apparent Km and V values obtained from these plots are given in Table IV. While no conclusion relative to the significance of these values may be drawn at this time, the values indicate interesting differences between the two tissues in response to increasing substrate concentrations. Phospholipase assays of host liver and Novikoff hepatoma microsomes using l-[ 9’,10’-3Hz]palmitoyl-2-[ 1’-14C]linoleoyl-sn-glycero-3-phosphoethanolamine in the presence of 1 mg of cyclic AMP or cyclic GMP showed no change in hydrolytic activities over controls containing equivalent amounts of 5’-AMP or 5’-GMP respectively. No differences were detected in the presence or absence of either calcium ions or Triton X-100 at pH 7.5 or pH 9.5. TABLE

IV

APPARENT LIVER

Km

AND

V VALUES

FOR

MICROSOMES

Liver

OF

NOVIKOFF

HEPATOMA

Hepatoma

PH

7.5

9.5

7.5

9.5

Km (PM) V (nmoI/mg/h)

44 * 51

100 250

54 217

125 500

* The numbers

are an average of four values obtained

in different

experiments.

AND

HOST

182

Discussion Lee and Snyder [ 50,511 have shown that specific phospholipid classes have similar turnover rates in endoplasmic reticulum and plasma membranes of rat liver and that the plasma membrane may be totally dependent on the endoplasmic reticulum for its supply of phospholipids. Wirtz [ 21 has proposed that specific proteins maintain a dynamic equilibrium of phospholipids between the endoplasmic reticulum and other cellular membranes. The microsomal fraction must therefore play a major role in the dynamics of membrane phospholipids. It has been pointed out [31] that phospholipase A activity may alter membrane permeability and the formation of membrane junctions by (a) altering the fatty acid composition of the membrane phospholipids, or (b) producing lysophospholipids that would induce a local micellar organization. Hax et al. [ 141 have suggested that cell communication is regulated by the combined activities of phospholipase A and acyltransferase under the control of divalent cations and possibly cyclic AMP [ 15,161. Because hepatoma cells lack the membrane junctions found in normal liver [ 521, it is of interest that the results of this paper show dissimilar phospholipase activities in the microsome of neoplastic liver cells. Host liver, regenerating liver and Novikoff hepatoma microsomes all show a calcium-dependent phospholipase Az that appears active on both 1-alkyl-2-acylsn-glycero-3-phosphoethanolamine and on phosphatidylethanolamine. Host liver and regenerating liver microsomes also show phospholipase A, activity that is stimulated by Triton X-100 and appears to be slightly inhibited by calciums ions. There is no evidence of any phospholipase Al activity in the hepatoma microsomes. There are also differences in the time course, the effects of increasing protein concentration and apparent Michaelis-Menten constants between the two tissues. Neither cyclic AMP nor cyclic GMP had any apparent effect on the hydrolysis of phosphatidylethanolamine. While the microsomes from all three tissues show lysophospholipase activity, the activity is inhibited by Triton X-100 to a greater extent in hepatoma then in host liver or regenerating liver. Since the overall phospholipase patterns in regenerating liver are similar to host liver, the differences in activities appear to be a property of the hepatoma rather than an effect of growth rate. Van den Bosch et al. [ 53-561 have purified to homogenity a detergent-sensitive lysophospholipase from beef liver microsomes which under certain incubation conditions shows a small amount of phospholipase A1 activity. They have also purified an enzyme from beef pancreas [57] whose specificity (A, vs. lysophospholipase) depends on the presence or absence of deoxycholate. In light of these observations in other tissues, further exploration of the nature of the differences in phospholipid hydrolytic activities of microsomes from normal and transformed rat liver will have to include purification of the phospholipases in order to determine whether the activities reside in multiple or a single protein. Such information may pave the way for studies that will increase our insight into differences in the structure and function of normal and neoplastic tissues.

183

Acknowledgement This investigation was supported by Public Health No. CA 13412 from the National Cancer Institute.

Service Research

Grant

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